Apparatus and methods for infrared calorimetric measurements

ABSTRACT

Apparatus and methods for performing calorimetry. The apparatus include optical devices for detecting thermal processes and multiwell sample plates for supporting samples for use with such optical devices. The methods include measurement strategies and data processing techniques for reducing noise in measurements of thermal processes. The apparatus and methods may be particularly suitable for extracting thermal data from small differential measurements made using an infrared camera and for monitoring chemical and physiological processes.

CROSS-REFERENCES

[0001] This application is based upon and claims benefit under 35 U.S.C.§119 of the following U.S. Provisional Patent Applications, each ofwhich is incorporated herein by reference: Serial No. 60/249,931, filedNov. 17, 2000; and Serial No. 60/256,852, filed Dec. 19, 2000.

[0002] This application is a continuation of U.S. patent applicationSer. No. ______, filed Jan. 17, 2001, titled APPARATUS AND METHODS FORINFRARED CALORIMETRIC MEASUREMENTS, and naming Andy C. Neilson, Jay S.Teich, Michael R. Sweeney, James D. Orrell III, Marc Samson, John M.Hopkins, and Michael W. Oster as inventors.

FIELD OF THE INVENTION

[0003] The invention relates to calorimetry. More particularly, theinvention relates to apparatus and methods for performing calorimetrythat use optical devices to detect thermal processes and/or multiwellsample plates to support samples for use with such optical devices.

BACKGROUND OF THE INVENTION

[0004] Thermodynamics has established the interrelationship betweenvarious forms of energy, including heat and work. Moreover,thermodynamics has quantified this interrelationship, showing, forexample, that in chemical and physiological processes the differencebetween the energy of the products and the energy of the reactants isequal to the heat gained or lost by the system. In an “exothermic”process, this difference is negative, so that the process releases heatto the environment. Conversely, in an “endothermic” process, thisdifference is positive, so that the process absorbs heat from theenvironment. Thus, “calorimetry,” or the measurement of heat productionand/or heat transfer, can be used to determine if a chemical orphysiological process is exothermic or endothermic and to estimate theenergy produced or consumed.

[0005] The measurement of heat production and/or heat transfer inchemical and physiological processes can be quite complicated.Standardly, such measurements are made using a device known as a “bombcalorimeter.” This device typically includes a sturdy steel containerwith a tight lid, immersed in a water bath and provided with electricalleads to detonate a reaction of interest inside the calorimeter. Theheat evolved in the reaction is determined by measuring the increase intemperature of the water bath.

[0006] Unfortunately, bomb calorimeters are inadequate for themeasurement of heat production and/or heat transfer in many areas ofchemistry and physiology. For example, the study of processes involvinguncommon and/or expensive components may require analysis of samples toosmall for bomb calorimetry. Similarly, the high-throughput screening ofpharmaceutical drug candidate libraries for drug activity may requireanalysis of too many samples for bomb calorimetry.

[0007] The analysis of small samples is especially problematic due totheir small heat capacities and large surface-to-volume ratios. Manychemical and physiological processes lead to very small changes intemperature (<0.05° C.), making their analysis susceptible toenvironmental contamination. In particular, whenever there is atemperature difference between a sample and the environment, heat can beexchanged between the sample and the environment, for example, byconduction, convection, and/or radiation, among others. Such heatexchange may quickly alter the temperature of a small sample and therebyobscure any temperature change associated with a reaction. Moreover,fluid samples such as those typically used in studies of chemical andphysiological processes may initiate secondary reactions with theenvironment, such as evaporation. Evaporation, by definition, is anexchange of energy (moisture is added to the air, while chemical volumeis reduced). This process takes place on the surface of the sample,where the sample is exposed to the environment, and so may be especiallyproblematic for small samples due to their relatively largesurface-to-volume ratios. Evaporation not only removes energy from thesample, contaminating the measurement, but also may increase measurementnoise due to surface instability as the fluid phase changes to a gasphase.

SUMMARY OF THE INVENTION

[0008] The invention provides apparatus and methods for performingcalorimetry. The apparatus include optical devices for detecting thermalprocesses and multiwell sample plates for supporting samples for usewith such optical devices. The methods include measurement strategiesand data processing techniques for reducing noise in measurements ofthermal processes. The apparatus and methods may be particularlysuitable for extracting thermal data from small differentialmeasurements made using an infrared camera and for monitoring chemicaland physiological processes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 is a partially schematic cross-sectional view of a systemfor detecting thermal processes.

[0010]FIG. 2 is a top view of a multiwell sample holder for use with anoptical device for detecting thermal processes.

[0011]FIG. 3 is a cross-sectional view of the multiwell sample holder ofFIG. 2, taken generally alone line 3-3 in FIG. 2.

[0012]FIG. 4 is a graph showing the infrared transmissivity of apreferred sample well material as a function of the thickness of thematerial.

[0013]FIG. 5 is a pseudo-color image showing the extent and effect ofthermal cross talk in sample wells in (A) the microplate of FIGS. 2 and3 and (B) a standard commercial microplate.

[0014]FIG. 6 is a graph showing noise envelopes associated withmeasurements of sample temperature taken from the (A) top and (B) bottomof a sample well after application of common-mode noise subtraction,area averaging, and frame averaging. The noise envelope associated withthe top-read data is significantly larger than the noise envelopeassociated with the bottom-read data due to evaporation.

[0015]FIG. 7 is a graph showing the size of the noise envelope as afunction of the number of frames average in an image-averagingexperiment.

[0016]FIG. 8 is a graph showing the effects of common-mode noise anddrift on thermal data collected using an infrared camera.

[0017]FIG. 9 is a graph showing the effects of offset subtraction onthermal data.

[0018]FIG. 10 is a graph showing the effects of removing common-modenoise on thermal data.

[0019]FIG. 11 is a software screen for use in the display of thermaldata, including temperatures and temperature differentials.

[0020]FIG. 12 is a software screen for use in the collection and/oranalysis of data from measurement and reference regions.

[0021]FIG. 13 is a software screen for use in defining selectedcharacteristics of the reference region.

DEFINITIONS

[0022] Technical terms used in this application have the meanings thatare commonly recognized by those skilled in the art. The following termsmay have additional meanings, as described below:

[0023] Common-mode noise. Typically low-frequency (<1 Hz) noise causedwhen internal control loops, such as the servo on a cryogenic cooler,create response changes in the detector. In an infrared camera, thesenoise sources may be common to each sensor element and may begeometrically displaced across the sensor array. For example, at a giventime, a thermal wave from the expansion of helium gas in a sensor coolermay cause slight gain changes in the sensor that cause a group of sensorelements in one geometric region of the array to respond differently, orout of phase with, another group of sensor elements in another region ofthe array. In most applications, common-mode noise is insignificant;however, in high-sensitivity (<0.05° C.) applications, common-mode noisemay become a limiting factor.

[0024] Heat. A form of energy associated with the motion of atoms ormolecules. Heat is capable of being transmitted by (1) conductionthrough solid and fluid media, (2) convection through fluid media, and(3) radiation through empty space.

[0025] Infrared (IR) radiation. Invisible electromagnetic radiationhaving wavelengths from about 700 nanometers, just longer than red inthe visible spectrum, to about 1 millimeter, just shorter than microwaveradiation. Infrared radiation includes (A) near IR (from about 700 nm toabout 1,300 nm), (B) middle IR (from about 1,300 nm to about 3,000 nm),and (C) far or thermal IR (from about 3,000 nm to about 1 mm). Near andmiddle IR is infrared radiation that typically is caused by vibrationsand low-level electronic transitions in molecules and that is onlyperipherally related to heat. In contrast, thermal IR (or thermalinfrared radiation) is infrared radiation that is caused or produced byheat and that is emitted by an object in proportion to the temperatureand emissivity of the object.

[0026] Radiosity. The radiation emanating from an object is determinedby the following parameters: (1) emissivity, i.e., the amount ofradiation the object emits, (2) reflectivity, i.e., the amount ofexternally derived radiation the object reflects, and (3)transmissivity, i.e., the amount of externally derived radiation theobject transmits. For example, the thermal power P radiated by an objectmay be described by the equation P=εσAT⁴, where ε is the emissivity ofthe object, σ is the Stefan-Boltzmann constant, A is the area of theobject, and T is the temperature. Emissivity, reflectivity, andtransmissivity are dimensionless parameters with values that rangebetween 0 and 1. For a given material, the sum of these parametersshould equal unity, so that each parameter is inversely correlated withthe sum of the other parameters. A material with an opaque surface has atransmissivity of zero, so its emissivity equals one minus itsreflectivity. Materials that radiate very well and absorb a largepercentage of the radiation that strikes them have high emissivities.

[0027] Parasitic noise. Typically low-frequency (<0.1 Hz) noise causedby stray radiation incident on the detector from within the detectorhousing, creating an offset in output that results in measurement error.The stray radiation may be caused by slight temperature changes internaland/or external to the detector. In an infrared camera, the error may begeometrically displaced across the camera array, as determined by theefficiency of the cold shield for the camera sensor and the bafflingwithin the Dewar. Most infrared cameras attempt to correct for parasiticnoise using some form of internal calibration mechanism, such as auniform-temperature shutter that periodically drops in front of thesensor to perform an offset compensation. These calibration mechanismsinherently interrupt measurements and can lead to measurement errors ifthe uniform-temperature shutter is not actually perfectly uniform intemperature. All infrared radiometers have some form of parasitic noise.

[0028] Spatial Noise. Typically lower-frequency (<60 Hz) highlynonlinear noise reflecting detector artifacts caused by variations inthe manufacturing process, for example, during metal oxide vapordeposition (MOVD). In an infrared camera, these artifacts may causeslight differences in the gain or response characteristics, spectralcharacteristics, and/or stability characteristics of the variouselements, columns, and/or rows of the sensor. Spatial noise may resultin low-frequency noise or a drift component, which may still remain evenafter performing a calibration for pixel gain and offset in the camera.

[0029] Temporal noise. Typically high-frequency (>60 Hz) random noisecaused by (radiated or conducted) electronic noise, A/D quantization,1/f noise, microphonics, and/or a low electronic signal-to-noise ratiofrom the detector.

[0030] Thermal conductivity. The quantity of heat transmitted, due to aunit temperature gradient, in unit time under steady conditions in adirection normal to a surface of unit area, when the heat transfer isdependent only on the temperature gradient.

[0031] Thermodynamic noise. Noise caused by thermodynamic instabilitiesin a medium, such as a fluid-to-gas phase transition. Surfacemeasurements of most fluids, including water, show significantinstability due to thermodynamic noise caused by evaporation.

DETAILED DESCRIPTION

[0032] The invention provides apparatus and methods for performingcalorimetry (or thermogenic analysis). The apparatus include opticaldevices for detecting thermal processes and multiwell sample plates forsupporting samples for use with such optical devices. The methodsinclude measurement strategies and data processing techniques forreducing noise in measurements of thermal processes. The apparatus andmethods may be particularly suitable for (1) extracting thermal datafrom small differential measurements made using an infrared camera, and(2) for monitoring chemical and physiological processes.

[0033]FIG. 1 shows a system 100 for detecting thermal processes inaccordance with aspects of the invention. The system includes an opticaldevice 102 configured to detect thermal radiation 104 and a sample plate106 having a sample well 107 configured to support a sample 108 for usewith the optical device. The system may be used to monitor thermalprocesses in the sample or samples by detecting temperature changescorrelated with heat production (e.g., from a chemical or physiologicalreaction) and/or heat transfer in the samples. This correlation may beperformed using any suitable method, such as those described in thefollowing U.S. provisional patent application, which is incorporatedherein by reference: Serial No. 60/256,852, filed Dec. 19, 2000. Ifthere are multiple samples, the radiation transmitted from the samplesmay be detected sequentially from each sample, for example, by pointreading, or simultaneously from some or all of the samples, for example,by image reading. The optical device may include a detector 110 such asan infrared optical sensor configured preferentially to detect thermalinfrared radiation. The detector measures thermal energy radiated from asample (or samples) supported by the sample plate and converts themeasured energy to a signal such as an electrical signal that can beconverted into a temperature, for example, using a blackbody or graybodyapproximation. In a preferred embodiment, the detector includes animaging device such as an infrared focal plane array (FPA) configured toobtain a time-dependent series of two-dimensional infrared images ofprocesses occurring in samples in a multiwell sample plate, permittingmeasurement of temperature and temperature changes in each process, as afunction of time, geometrically across the plate. The series of imagestypically is collected at a preselected frequency (typically >1 Hz) fora preselected period significant relative to a characteristic time ofany time-dependent process being monitored. The data subsequently may beprocessed and/or reported at a lower frequency. The data may be used tomonitor, screen, rank, and/or otherwise analyze thermal processesoccurring in the sample. The thermal analysis may be used alone ortogether with other measurements to assess the presence, concentration,physical properties, and/or activity of a compound or compounds in thesample. Thus, the system provides a noncontact, noninvasive method formeasuring thermal properties such as temperature, in contrast to bombcalorimeters, thermometers, and capacitive and resistive circuits.

[0034] The system and its components may be configured to improve theaccuracy and/or sensitivity of thermal measurements, particularlythermal measurements involving small samples and/or small temperaturechanges. The optical device may be configured to reduce measurementerrors associated with noise, such as common-mode, parasitic, spatial,temporal, and/or thermodynamic noise, among others. The sample plate maybe configured to facilitate detection of thermal radiation through asurface of the plate and/or to reduce measurement-contaminating heattransfer between the samples and the environment (including othersamples). The optical device and sample plate may together be configuredto reduce noise associated with evaporation, for example, by using abottom-read detector and a sample plate having an infrared-transmissivebottom surface and in some cases a cover.

[0035] The remainder of the Detailed Description is divided into foursections: (A) optical devices, (B) noise reduction, (C) sample holders,and (D) examples.

A. Optical Devices

[0036] The optical device generally comprises any device capable ofpreferentially detecting thermal infrared radiation and using thedetected radiation to analyze thermal processes in a sample. The phrase“capable of preferentially detecting thermal infrared radiation” meansthat the device is configured and/or operated so that it detects morethermal infrared radiation than any other form of radiation (i.e., sothat at least about half of the radiation detected is thermal infraredradiation). The phrase excludes any device that detects thermal infraredradiation only incidentally, as might occur in an optical deviceconfigured to detect visible light if thermal radiation leaked into thedetector. The capability for preferentially detecting thermal infraredradiation may reflect use of one or more of the following mechanisms,among others: (A) use of spectral filters preferentially to “extract”thermal infrared radiating by blocking radiation other than thermalinfrared radiation, including visible, near IR, or middle IR radiation,(B) use of detectors having enhanced sensitivity for thermal infraredradiation, and/or (C) postprocessing of a detector signal to reduceand/or compensate for any component of the signal not resulting fromdetection of thermal infrared radiation.

[0037]FIG. 1 shows an optical device 102 constructed in accordance withaspects of the invention in use as a part of a system 100 for detectingthermal processes. The device includes a detector 110 configured todetect thermal infrared radiation emitted by a sample, a stage 112configured to support a sample in a sample holder for thermal analysisby the detector, and a processor 114 configured to analyze radiationdetected by the detector. The detector and stage are positioned suchthat at least a portion of the thermal infrared radiation emitted by thesample is incident, indirectly or preferably directly, on the detector.This may be accomplished by ensuring that a central axis CA of thesample wells is aligned with an optical axis OA of the instrument priorto detection of thermal infrared radiation. Alignment simply means thatthe two axes are sufficiently close to parallel that radiation from acentral portion of the sample well is detectable by the instrument. Forexample, in FIG. 1, the central axis of each sample well is aligned withthe optical axis of the instrument. The detector may be positioned belowthe stage to form a “bottom-read” instrument, above the stage to form a“top-read” instrument, or in other positions to form other instruments.The instrument of FIG. 1 is a bottom-read instrument, and the instrumentof FIG. 1 with components of the optical device 102 inverted andpositioned above the stage is a top-read instrument.

[0038] The stage may be movable, so that samples may be deposited at afirst position, moved to a second position for fluid dispensing, movedto a third position for thermal equilibration, moved to a fourthposition for thermal detection, and moved to a fifth position forpickup. These positions may be the same or different, and any givenposition (except the detection position) may be present or absent. Thestage may move sample holders translationally and/or rotationally, amongothers.

[0039] The sample generally comprises any object or system of objectsintended for thermal analysis. The sample may include compounds,mixtures, surfaces, solutions, emulsions, suspensions, cell cultures,fermentation cultures, cells, tissues, secretions, and/or derivativesand/or extracts thereof. The sample also may include the contents of asingle sample site, or several sample sites, depending on the assay.

[0040] The detector generally comprises any device for preferentiallydetecting thermal infrared radiation and converting the detectedradiation into a signal representative of the detected radiation. Apreferred detector is an imaging detector, such as an infrared camera,that is capable of simultaneously viewing part or all of a sampleholder.

[0041] The stage generally comprises any mechanism for supporting asample in a sample holder at an examination site for thermal analysis bythe device. A preferred stage is a transporter capable of moving thesample holder vertically and/or horizontally between the examinationsite and one or more transfer sites where the sample holder can beloaded onto and/or unloaded from the stage.

[0042] The processor generally comprises any mechanism for analyzing thesignal from the detector, for example, to conduct a thermal analysis.The analysis may include conversion of a signal representative ofintensity and/or wavelength into a signal representative of temperatureand/or differential temperature, among others. The analysis also mayinclude performing calculations to reduce noise and/or to facilitatedata reporting, as described below. The processor may be intrinsic tothe detector, extrinsic to the detector, or both.

[0043] The optical device also may include a housing 116 to support andprotect the detector. The housing may include, among others, an opticstube 118, an infrared-transmissive window 120, and/or a baffle 122having an aperture 124. The optics tube may support the detector and/orcomponents of the housing, such as the window and baffles. The opticstube also may reduce the amount of unintended thermal infrared radiationentering the detector. The window may permit thermal infrared radiationto enter the housing for detection, while also permitting the housing tobe sealed to reduce contamination and/or (partially) evacuated to reduceabsorption and/or scattering of thermal radiation prior to detection.The window may include a portion formed of zinc selenide (ZnSe) and/orpolyethylene, among others. The baffles may block stray thermal infraredradiation. The optical device may be configured to shield the samplefrom incident radiation to reduce the proportion of the sample signalarising from transmission, reflection, and/or photoluminescence from thesample.

[0044] The optical device also may include a data output mechanism suchas a view screen or printer for reporting results of any thermalanalysis. Data generally may be reported using any suitable method,physical and/or electronic, digital and/or analog, and static and/ortime varying, among others. Suitable methods include tables, graphs,and/or images, among others. Data may include temperatures and/ortemperature differentials, among others, at a fixed time or as afunction of time.

B. Noise Reduction

[0045] Noise is almost invariably a problem in measurements of thermalprocesses. However, noise may be especially problematic in measurementsof thermal processes involving small samples and/or small temperaturechanges, where even minor noise can obscure or overwhelm any temperaturechange associated with the thermal process.

[0046] The effects of noise on measurements of thermal processes can bereduced using noise reduction techniques. Noise reduction involvesidentifying sources of noise (e.g., measurement noise, camera noise,etc.) and then developing methods for reducing or eliminating the noiseor its effects. Unfortunately, thermal detectors such as infraredcameras are susceptible to several types of noise, includingcommon-mode, parasitic, spatial, and temporal noise, among others. Inaddition, fluid samples are susceptible to other types of noise,including thermodynamic noise, which is caused by thermodynamictransformations, such as evaporation, in which the sample changes from afluid to a gas. Thus, noise reduction in measurements of thermalprocesses may involve the application of one or more different methods.

[0047] The present “state of the art” for infrared, radiometric camerasdefines measurement performance in terms of noise-related parameters,specifically, accuracy and sensitivity. Here, accuracy is a relativeabsence of error or mistake, and sensitivity is an ability to detect ormeasure an input, especially a weak input. The sensitivity of infraredcameras normally is quantified in terms of noise equivalent temperaturedifference (NETD), which is the RMS noise/response at a giventemperature, f#, and operating frequency (normally 30° C., f/1, and 60Hz). NETD typically is the limiting factor in determining measurementsensitivity. A typical, state-of-the-art, high-performance infraredcamera, such as the FLIR SC3000, using Quantum Well (QWIP) sensortechnology, specifies an absolute accuracy of about 2° C. and asensitivity (NETD) of about 0.03° C. Unfortunately, this sensitivity maybe inadequate in measurements of small temperature changes.

[0048] The invention provides methods for reducing noise and/orenhancing accuracy and/or sensitivity in thermal measurements,particularly measurements involving small samples and/or smalltemperature changes. These methods may be implemented using any suitableapparatus, such as any processor associated intrinsically orextrinsically with the optical device. These methods may improve uponthe previous state-of-the-art sensitivity described above, potentiallyproviding sensitivities of <0.01° C. and RMS noise levels of <0.005° C.,at least when the methods are used on data collected with preferredsample holders. The methods may involve application of one or more ofthe following techniques, among others:

[0049] 1. Low-pass Filtering

[0050] Data may be collected at a relatively high frame rate and passedthrough a low-pass time-domain) filter to reduce high-frequency“temporal noise.” For example, using an infrared camera as describedabove, full-field radiometric data may be collected at a 60 Hz framerate and passed through a low-pass filter internal to the cameraelectronics to yield filtered data corresponding to a lower frame rate.

[0051] 2. Frame Averaging

[0052] Data may be averaged (or otherwise smoothed) over a series offrames to remove any residual high-frequency temporal noise. Forexample, frame averaging may be performed pixel-by-pixel by summing thevalues T_(ij) associated with a given pixel ij in each frame k and thendividing the sum by the total number N of frames used in the average:$\begin{matrix}{{\langle T_{ij}\rangle}_{FA} = {\frac{1}{N}{\sum\limits_{k = 1}^{N}{T_{ij}(k)}}}} & (1)\end{matrix}$

[0053] Here, <> denotes averaging. Typically, the middle frame in theset of N frames is replaced by the average frame. The preferred numberof frames to use in the frame average is determined by competingfactors. Generally, it is better to use a larger number of framesbecause frame averaging and low-pass filtering typically reducehigh-frequency random temporal noise by the square root of the number offrames averaged. However, the overall time corresponding to the numberof frames used in the average should be small relative to the time scaleof thermal changes in the system to avoid averaging frames that differdue to actual differences in the temperature of the sample rather thandue merely to noise. In the data presented below under Examples, theoptimum number of frames for frame averaging was between about 4 and 16.Frame averaging may be performed separately for measurement andreference areas.

[0054] 3. Area Averaging

[0055] Data may be spatially averaged to return a reduced number of(average) values or a single (average) value for each area as a functionof time. For example, area averaging may be performed by summing thevalues T_(ij) associated with some or all of the pixels in a given areaA of a frame and then dividing the sum by the total number M of pixelsused in the average: $\begin{matrix}{{{\langle T\rangle}_{AA}(k)} = {\frac{1}{M}{\sum\limits_{i,{j\quad \in \quad A}}{T_{ij}(k)}}}} & (2)\end{matrix}$

[0056] Thus, in a sample holder having 96 sample wells each having ameasurement area and a reference area, area averaging may be used toreduce the data set to as few as 96 measurement values and 96 referencevalues by independently averaging pixel values over all or part of eachmeasurement and reference area. The measurement and reference areas maybe distinguished using application software implemented in theprocessor. Area averaging typically involves >4 pixel elements andpreferably involves >9 pixel elements. Area averaging may reduce theeffects of geometric, spatial noise common to most FPA detectors. Suchnoise may reflect defective or nonlinear detector elements and/or slightdifferences in amplifier characteristics.

[0057] 4. Reference Calibration

[0058] Data may be calibrated using a reference standard, such as anadjacent local (e.g., perimeter) reference standard, for example, bysubtracting a reference value from a corresponding measurement value toreturn a differential measurement for each sample well as a function oftime.

T _(RC) =T _(Meas) −T _(Ref)  (3)

[0059] Here, the measured and reference values may be properties of thethermal radiation detected from the measurement and reference regions,respectively, such as intensities, or they may be quantities derivedfrom such properties, such as temperatures. The method may be appliedpixel-by-pixel or area-by-area, among others. Subtracting referencevalues from measurement values may reduce or eliminate common-modenoise, internal drift, and/or parasitic noise local to the region of thedetector array used in the measurements. These noise sources have atendency to be geometrically dispersed across the sample holder orsample wells, so that other noise-reduction techniques, such assingle-point reference or Fourier transform characterization andsubtraction have limited success. These other methods have a tendency toamplify noise where it shifts out of phase relative to adjacent areas,whereas the local reference compensates for geometric shifting.

[0060] 5. Offset Subtraction

[0061] Data may be adjusted by subtracting one or more offsets from eachmeasurement.

T _(OS) =T−T _(Offset)  (4)

[0062] The offset may be used to set the initial-time differentialmeasurements for each sample well at t (time)=0, so that there is acommon starting point from which to measure changes in temperature.Offset subtraction effectively creates a zero reference at the beginningof the experiment and adjusts the difference in temperature between themeasurement region and associated reference region to zero. Adjustingthe offset to zero may compensate for field nonuniformity resulting fromcamera drift prior to the start of data collection.

[0063] 6. Bottom Reading

[0064] Reading through the bottom of an infrared-transmissive samplewell may reduce thermodynamic noise created at the interface of dry airand the sample. In particular, evaporation at sample surfaces exposed todry air may create a saturated gas layer adjacent the sample surface.This layer may be opaque or nearly opaque to the thermal detector andshow significant instability (measured to be >0.05° C.). Measurementnoise created by evaporation may be fivefold or more greater thanmeasurement noise associated with reading through the bottom of thesample well or from an independent black body reference. Additionally,evaporation at the surface may lower the surface temperatures measuredby the camera by as much as 2° C. This 2° C. difference is a heat sinkfor the reaction being measured. Bottom reading allows the top surfaceof the sample well to be sealed so that the space above the samplebecomes saturated with moisture, reducing evaporation noise and heatloss.

[0065] The application of these noise-reduction methods generally isquite flexible. For example, each method generally may be appliedseparately, alone or in combination with any number of other methods.Moreover, each method generally may be applied in any order.

B. Sample Holders

[0066] The sample holder or sample plate generally comprises anysubstrate or material capable of supporting a sample for thermalanalysis. Suitable sample holders may include microplates, PCR plates,biochips, chromatography plates, and microscope slides, among others,where microplate wells and biochip array sites comprise assay ormeasurement sites.

[0067] The sample holder may include a thermal isolation structuredisposed between the sample wells to reduce thermal transfer between thewells and the environment and thus between adjacent wells. The thermalisolation structure may include a thermal buffer, thermal barrier,and/or isolation well, among others, as described below. The thermalisolation structure may be composed at least in part of a differentmaterial than the sample wells. The thermal isolation structure maysubstantially surround a central or optical axis of each sample well,isolating the wells without obstructing transmission of thermal infraredradiation along the central axis. The thermal isolation structure alsomay be disposed such that any straight line below a plane formed by thetops of the sample wells connecting a portion of one sample well to aportion of an adjacent sample well intersects the isolation structure.

[0068] The sample holder also may include an insert member defining anarray of sample wells and a support member having a thermal isolationframework in a configuration corresponding to the array of sample wells.The sample wells each may have a central axis, and the insert may engagethe support member such that each sample well is thermally isolated fromadjacent sample wells without obstructing the transmission of thermalinfrared radiation along the central axis. The thermal isolationframework may include a thermal buffer, thermal barrier, and/orisolation well, among others, as described below.

[0069] The sample holder also may include an insert having a pluralityof sample wells, and a thermal isolation member for supporting theinsert so that each sample well can be precisely positioned along anoptical path, where the thermal isolation member provides a thermallycontrolled thermal reference surface adjacent each well as viewed alongthe optical path. The reference surface may define an aperture thatframes the associated optical path.

[0070] A preferred sample holder is configured as a microplate having aframe and a plurality of sample wells disposed in the frame for holdinga corresponding plurality of samples for analysis. This format maycombine small-volume samples and a high-density holder, permittingautomated analysis of large numbers of samples. This format also may beconfigured to reduce unintended heat exchange between the samples andthe environment (including between the sample and other samples) and/orto permit an optical detector to measure thermal infrared radiationtransmitted through a surface of the sample holder.

[0071] The sample holder may include one or more of the followingfeatures, among others:

[0072] 1. Thin Surface

[0073] A sample well having at least one surface having a thickness ofless than about 0.005 inches, and preferably less than about 0.001inches, and most preferably less than about 0.0005 inches. A thinsurface may be important for at least two reasons: (1) increasedinfrared transmissivity, and (2) decreased thermal conductivity. Thesetwo criteria preferably may be met using a single material, such as apolymeric polyethylene blend having a high infrared transmissivity(e.g., greater than about 50% or about 80%) and a low thermalconductivity (e.g., less than about 1 W/m-K or about 0.6 W/m-K).

[0074] A thin (i.e., reduced-thickness) surface may increasetransmissivity. A thin surface may be less likely to absorb thermalenergy being radiated by the sample due to its shorter path length andmore likely to have an outer (i.e., non-sample-contacting) surface atthe same temperature as the sample, facilitating calorimetric analysisthrough the surface. A preferred thin surface has a high transmissivity(e.g., >80%) for thermal infrared radiation, particularly thermalinfrared radiation having wavelengths between about 3 and 5 micrometersand between about 7 and 14 micrometers. (These wavelength ranges may beespecially useful in thermal imaging, because they correspond to minimain atmospheric absorption.) A thin more transmissive surface preferablyis located at least at the bottom of the sample well to permit detectionfrom the underside of the sample holder using a bottom-read analyzer.The surface may be substantially (e.g., optically) flat to reduceoptical aberrations during analysis through the surface.

[0075] A thin (i.e., reduced-thickness) surface also may decreasethermal conductivity. There are three primary mechanisms for heattransfer in the plate: conduction, convection, and radiation. Typically,conduction is the most significant mechanism, and radiation is the leastsignificant mechanism. Conduction may be described by the equationP=KA∇T, where K is the thermal conductivity, A is the surface area, and∇T is the temperature gradient. Thus, reducing surface area may reduceconduction. A primary path for conduction is through the walls of thesample well to contact points on the associated frame. This path may bereduced using thin-walled sample wells. Moreover, because the thermalconductivity of air (˜0.02 W/m-K) is less than that of the preferredwell material (0.6 W/m-K), it is important to use the air as much aspossible for a conduction path. Thus, the wells hold heat much like athermos. Finally, the thermal capacitance of a thin material is lower,so that there is less change in temperature due to the initial ΔT in thesystem. In particular, the material may be selected such that thethermal mass of the sample wells is no more than about half the thermalmass of an aqueous sample positioned in the sample well, even when thesample well is completely full. A thin less conductive surfacepreferably is located at least at the sides of the sample well.

[0076] 2. Thermal Buffer

[0077] A thermal buffer disposed between the sample wells to resistthermal transfer between sample wells, or between the environment andthe sample wells. The thermal buffer generally comprises any mechanismfor resisting a change in temperature. In this sense, the thermal bufferresembles a pH buffer, which resists a change in pH when an acid or baseis added to a solution by binding to the added species, or an electricalcapacitor, which resists a change in voltage by storing or releasingcharge. The thermal buffer may be used to buffer (or keep relativelyconstant) the temperature of any structure adjacent the sample well,such as the trapped volume described below. The thermal buffer mayinclude a structure having a high thermal mass (or heat capacity), whichcan absorb heat without undergoing a significant change in temperature.This high thermal mass structure may, for example, have a substantiallyhigher thermal mass (or heat capacity) than the sample wells and/orcorresponding samples, for example, three, five, or even ten timeshigher. The high thermal mass structure may include a metal such asaluminum and/or a high thermal capacitance plastic, among others.

[0078] 3. Thermal Barrier

[0079] A thermal barrier disposed between the sample wells to blockthermal transfer between sample wells. The thermal barrier generallycomprises any mechanism for blocking the transfer of heat into or out ofthe sample wells or the vicinity of the sample wells, such as anadjacent trapped volume. The thermal barrier may include a materialhaving a low emissivity and/or a high reflectivity for infraredradiation. For example, the thermal barrier may include a material thatreflects at least about half of the infrared radiation that otherwisewould be incident upon surfaces of the sample well. Generally,emissivity and reflectivity are inversely related; thus, shiny, metallicmaterials tend to have low emissivities and high reflectivities, whereasmatte, dark-colored materials tend to have high emissivities and lowreflectivities. In a preferred embodiment, the thermal barrier includesa material having a reflectivity of at least about 0.8 and an emissivityof at most about 0.2.

[0080]4. Double-walled Sample Wells

[0081] A double-walled sample well, formed, for example, by positioninga sample well in a corresponding isolation well. In a preferredembodiment, a plurality of isolation wells are disposed in a frame, acorresponding plurality of sample wells are disposed in the isolationwells, and none of the sample or isolation wells is in fluid contactwith another of the sample or isolation wells. The double-walled wellsmay include a trapped volume formed between an outer surface of thesample wells and an inner surface of the corresponding isolation wells,further reducing thermal transfer to and from samples positioned in thesample wells. The trapped volume may enclose air and/or an inert gas,and/or be partially or fully evacuated relative to standard atmosphericpressure. The trapped volume also may enclose or be lined along itsperimeter with a thermal barrier, i.e., a material having a lowemissivity and/or a high reflectivity for infrared radiation to reduceradiation thermal transfer to and from the sample well.

[0082] 5. Plural Optically Transmissive Surfaces

[0083] A plurality of optically transmissive surfaces, at least oneassociated with the frame and at least one associated with the samplewell, where the surfaces are configured so that an optical reader candetect electromagnetic radiation such as infrared radiation transmittedfrom a sample through both the optically transmissive surface of thecorresponding sample well and the optically transmissive surface of theframe. For example, a plurality of optically transmissive surfaces maybe formed by corresponding surfaces of a sample well and isolation wellin a double-walled well, as described above.

[0084] 6. Measurement and Reference Regions

[0085] A combination of a measurement region and a reference region. Themeasurement region may be a portion of a sample well, and the referenceregion may be an adjacent portion of the frame, isolated from the samplewell, particularly a high-thermal-mass and/or high emissivity (>0.5 andpreferably >0.8) surface portion capable of acting as a blackbody orgraybody reference. The reference region may be composed at least inpart of a different material than the sample wells and may include ametal such as aluminum. The reference region(s) may be disposed adjacent(e.g., about or between) the sample wells, so that each measurementregion is near a corresponding reference region, reducing artifacts thatreflect temperature drift across the sample plate. Thus, an M×N array ofmeasurement regions might be complemented by an M×N of reference regionsdisposed about the measurement regions, or an (M−1)×(N−1) array ofreference regions disposed between the measurement regions. Thereference region may be configured as a ring or annulus distributedabout or adjacent a perimeter of the sample well and/or about andpreferably symmetrically about a central axis of the sample well. Thereference region may be positioned about or above the top of acorresponding sample well, and/or about or below a corresponding samplewell. The reference regions and the corresponding sample wells may beseparated by a gap such as an air gap along a line connecting eachportion of the thermal reference regions and the corresponding samplewells to reduce heat transfer between the sample wells and the thermalreference regions. Thermal characteristics of the measurement region maybe calibrated using thermal characteristics of the reference region, forexample, by subtracting the reference characteristic from themeasurement characteristic. This calibration may reduce geometricallydispersed common-mode noise, including the effects of internal parasiticradiation and camera drift. The use of dedicated reference regions mayfree up all of the sample wells for data analysis, because none of thewells needs to be used as a reference well.

[0086] 7. Consumable Sample Well Inserts

[0087] A combination of a reusable frame and a consumable sample wellinsert (or a set of consumable sample well inserts) configured to fitwithin or mate with the frame. The combination may facilitate reuse ofportions of the sample holder that are expensive, such as the thermalbuffer and/or thermal barrier. The combination also may facilitatedisposal of portions of the sample holder that contact the sample byreducing the amount of such materials that must be discarded. Thecombination may be constructed so that the insert is substantiallysupported by the frame yet substantially thermally insulated or isolatedfrom the frame. The frame and the sample wells may be composed of thesame or preferably different materials. Here, consumable may be definedas more likely to be discarded than reused, typically because it is moreconvenient and/or less expensive to be discarded than reused. Forexample, a consumable sample well insert may obviate the need to cleansample wells between samples.

[0088] 8. Cover

[0089] A cover configured for use with the sample holder. The covergenerally comprises any mechanism for covering the sample holder, or aportion of the sample holder, to reduce contamination of the samplesand/or to reduce evaporation from the samples (e.g., by reducingexposure to dry air and/or convective air currents), among others. Thecover generally may be formed of any suitable material, such as a rigidplastic and/or a thin layer of oil or other less evaporative materiallayered over the sample. The cover may be infrared transmissive, so thatsamples may be analyzed through the cover using a top-read analyzer. Thecover may be configured to touch the top surface of the sample.Alternatively, the cover may be configured to leave an air gap betweenthe top surface and the cover, particularly a small air gap that mayquickly saturate with fluid vapor after fluid samples are positioned inthe wells and before reactant or catalyst are delivered to reduceevaporative cooling during analysis. Generally, evaporation may bereduced by reducing the size of the air gap, for example, by usingshallow wells and/or by substantially filling the wells (for example,until the samples occupy at least about half or even about eight-tenthsor nine-tenths of the volume of the sample wells). Alternatively, or inaddition, evaporation may be reduced by increasing the humidity of theair adjacent the sample well or sample holder. The cover may include anaperture so that a fluid delivery system such as a pipette can piercethe cover and deliver reactant fluids.

D. EXAMPLES

[0090] The following examples describe without limitation furtheraspects of the invention. These examples show that thermal cross talkbetween sample wells can be reduced by thermally isolating the samplewells and that thermal resolution and the accuracy and sensitivity ofthermal measurements can be enhanced by reducing noise, includingcommon-mode, parasitic, spatial, temporal, and/or thermodynamic noise,among others. Additional examples including color drawings showingpseudocolor methods for displaying thermal imaging data are described inthe following U.S. provisional patent application, which is incorporatedherein by reference: Serial No. 60/256,852, filed Dec. 19, 2000.

Example 1

[0091] This example describes a preferred sample holder for use inmeasurements of thermal processes, including chemical and physiologicalprocesses.

[0092]FIGS. 2 and 3 show a sample holder 200 constructed in accordancewith aspects of the invention. The sample holder includes a high thermalmass frame or base 202, a plurality of sample wells 204 and acorresponding plurality of windows 206, trapped volumes 208, referenceregions 209, and opaque coatings 210, and a cover 212.

[0093] Frame 202 is the main structural component of sample holder 200.The frame generally may be sized and shaped as desired, for bothconvenience and utility. Frame 202 is sized and shaped to form amicroplate, enabling the sample holder to be used with standardmicroplate equipment, such as handlers, washers, and/or readers, amongothers. A preferred frame is substantially rectangular, with a majordimension X of about 125-130 mm, a minor dimension Y of about 80-90 mm,and a height Z of about 5-15 mm, although other dimensions are possible.Frame 202 may include a base 214 configured to facilitate handlingand/or stacking, a notch 216 configured to facilitate receiving thecover, and/or a plurality of apertures 218 configured to receive andsupport a corresponding plurality of sample wells. The apertures provideclearance around the sample wells, creating an air gap that may providethermal isolation between the base and the sample well. The aperturesmay be formed using any suitable method, including machining and/orcasting the frame to include the apertures. The inner surface of eachaperture may be polished and/or lined with an opaque (i.e., lowtransmissivity) coating 210 such as AlSiO or gold to reflect infraredradiation and thus to form a thermal barrier to heat conduction to andfrom the sample wells. In this embodiment, adjacent sample wells may beseparated by two thermal barriers and a portion of the frame disposedbetween the two thermal barriers. The apertures and/or the sample wellsmay be tapered, such that the separation between the sample wells andthe walls of the corresponding apertures increases from the top to thebottom of the sample wells, further reducing conduction between thesample wells and the walls of the apertures.

[0094] The frame generally may be constructed of any suitable material.For example, frame 202 is constructed using a material having a highthermal mass (heat capacity) and high thermal conductivity, such asaluminum and/or other metals. Preferred materials such as aluminum mayreduce the time required for thermal stabilization within the testchamber while being sturdy enough for repeated, rugged use. Inparticular, a high thermal mass base (and/or an adjacent structure) mayfunction as a thermal buffer, helping to maintain a constant temperaturearound sample wells positioned in apertures 218.

[0095] Sample wells 204 are used to support and separate samples 220 forcalorimetric analysis. These sample wells may vary in size, shape,number, and arrangement, generally as desired, as long as the wells fitin the frame, and more particularly fit within the correspondingapertures in the frame. Exemplary sizes range between about 1 μL andabout 500 μL, and more preferably between about 1 μL and about 200 μL.Exemplary shapes include cones, frustums of cones, cylinders, andparallelepipeds, among others. Exemplary numbers include 96, 384, 864,1536, 3456, and 9600, among others. Exemplary arrangements includerectangular and hexagonal arrays, among others. Three preferredsample-well configurations that will fit as rectangular arrays within amicroplate-sized frame are listed in the following table: NumberArrangement Pitch (mm) Density (/mm²) of Wells of Wells Between Wells ofWells 96  8 × 12 9  1/81 384 16 × 24 4.5  4/81 1536 32 × 48 2.25 16/81

[0096] Here, pitch is the center-to-center well-to-well spacing, anddensity is the number of wells per unit area. In a preferred embodiment,the frame will include a similarly spaced array of apertures forreceiving the sample wells. A preferred configuration includes 96frustoconical wells organized in an 8×12 rectangular array. Here,frustoconical refers to a well shape having conical sides and a flatbottom, as shown in FIG. 3.

[0097] The sample wells may be formed as cups that may be inserted intothe frame to hold a sample for calorimetric analysis, permitting ananalyzer to measure temperature changes in the sample, for example,resulting from chemical or physiological processes. In this way, asingle frame may be used to support cups of different sizes and shapes,so long as the cup (or cups) will fit within the correspondingapertures. The bottom surface 221 of the cup may be flat and/orparticularly thin in areas from which infrared measurements arecollected. A flat surface may serve to reduce optical distortion andcontrol reflections coming from the surface. A thin surface may enhanceinfrared transmission, because infrared properties generally areproportional to material thickness. A thin surface also may help toensure that the outer surface remains at or very close to thetemperature of the fluid.

[0098] The cup inserts may be formed individually or joined to form asheet or sheets of cups for use in the frame. Individual cups and smallsheets of cups generally provide greater flexibility, permitting cups tobe mixed and matched (e.g., according to size, shape, and/or infraredtransmission properties, among others) within a single plate. Largesheets of cups provide greater structural stability and convenience,permitting many cups to be changed at once. As mentioned above, cupinserts generally are configured so that a single cup resides within asingle aperture in the supporting frame. However, cup inserts also maybe configured so that two or more cups reside within a single aperture,permitting a single frame to support cups at two or more significantlydifferent sizes and/or densities.

[0099] The material properties of the cups are important for thermalisolation and infrared transmissivity. FIG. 4 shows the infraredtransmissivity of a preferred cup material as a function of materialthickness. This preferred material is an infrared-transmissive(polymeric) polyethylene blend sold under the trademark Poly IR 2™ byFresnel Technologies. The material has a high infrared transmissivitythat increases nonlinearly with decreasing material thickness, showing asignificant increase for thicknesses below about 0.001 inches. Moreover,the material has a low coefficient of thermal conductivity (˜0.6watts/meter-kelvin), which is about {fraction (1/10)} the thermalconductivity of most other infrared-transmitting materials, includingZn, Se, and Ge. In addition, the material has a low thermal mass, so itshould quickly assume the temperature of the sample. The low thermalmass of the cup combined with its low thermal conductivity andinsulation from the base reduce heat loss to the environment. Theinfrared transmission properties of the material allow the detector tomeasure the temperature of the fluid through the cup with less than 10%thermal contribution from the cup. Further aspects of the preferredsample well material are described in the following U.S. provisionalpatent application, which is incorporated herein by reference: SerialNo. 60/256,852, filed Dec. 19, 2000.

[0100] Window 206 is an environmental seal between the sample well andthe detector(located below the sample holder) when the sample holder isused with a bottom-read analyzer. The window may be formed of aninfrared-transmissive membrane material selected to enhance infraredtransmission within the spectral sensitivity band of the camera. Apreferred material is zinc selenide, which provides >97% transmission tothe bottom surface of the sample well.

[0101] Trapped volume 208 is formed between inner surfaces of frame 202,sample well 204, and window 206. The high thermal mass frame thatsurrounds the sample well acts as a capacitor to maintain a constanttemperature within the trapped volume, which typically contains air. Theinner surfaces of the frame may be lined with an opaque coating, asdescribed above.

[0102] Reference region 209 is a source of a reference signal for use inreference calibrations to reduce common-mode and parasitic noise, amongothers, as described above. Generally, each sample well includes ameasurement region and a corresponding reference region. The measurementregion generally comprises a portion of the sample or sample well, suchas an infrared transmissive bottom portion of the sample well for usewith bottom-read instruments. The reference region may comprise anadjacent portion of the frame, such as an annular donut-shaped portionformed around a perimeter and/or central axis of the measurement region.Here, the reference region is positioned at an end of a support memberformed by portions of the frame disposed between the sample wells. Thethermal mass of the thermal reference region and associated supportmember may be at least about the same as or greater than the thermalmass of the corresponding sample well and/or sample. The referenceregion may be formed of a high thermal mass and/or high (>0.8)emissivity material such as a metal that acts as an isolated, blackbodyreference. The thermal reference region may include a substantially flatemissive reference surface, where the emissive surface is substantiallyparallel to a flat portion of the bottom of the sample well and/or wherethe emissive surface is within a factor of ten of the area of a flatportion of the bottom of the sample well.

[0103] Cover 212 provides a mechanism for covering the sample holder, ora portion of the sample holder, to protect samples from evaporationand/or reduce the likelihood and amount of evaporation. Cover 212generally will leave a small air gap between the sample and the coverthat may saturate with fluid vapor to reduce evaporation. Cover includesan aperture 222 so that a fluid delivery system such as a pipette canpierce the cover and deliver reactant fluids.

Example 2

[0104]FIG. 5 shows results from an experiment designed to measurethermal cross talk caused by heat conduction through the sample plate.The experiment was performed using a top-read thermal-imaging apparatusfitted with a quantum well (QWIP) infrared radiometer from FLIR Systems.The figure shows thermal images of two plates containing a fluid thatevaporates when in contact with dry ambient air. Here, relativetemperature is denoted by shading, where samples with relatively hightemperatures have increased shading, and samples with relatively lowtemperatures have reduced shading. Plate 1 (left) is fabricated using athin polymer insert and a high thermal mass base, as shown in FIGS. 2and 3. Plate 2 (right) is a standard commercially available 96-wellmicroplate fabricated from a polystyrene polymer (Costar 3628). Thethermal images show that plate 1 provides significantly better thermalisolation than plate 2. In particular, the wells in plate 1 areinsulated from the surrounding base material, reducing thermal “crosstalk” between adjacent wells, whereas the wells in plate 2 are poorlyinsulated, enhancing thermal “cross talk” and leading to significantthermal gradients across the plate.

Example 3

[0105] This example describes results of an experiment designed to testthe effects of evaporation on the apparent temperature of samplespositioned in wells in a multiwell plate.

[0106] The experiment was performed using the top-read thermal-imagingapparatus and low cross-talk multiwell plate of Example 2. In theseexperiments, sets of adjacent wells were filled with fluids havingvarious evaporation characteristics or else were covered with ahigh-emissivity tape, as shown below. T T T T T T T T T T T T T T T W WW A A A T T T T T T W W W A A A T T T T T T W W W A A A T T T T T T O OO W W W T T T T T T O O O W W W T T T T T T O O O W W W T T T T T T T TT T T T T T T

[0107] Here, A=alcohol, W=water, O=mineral oil, and T=tape. Alcohol andwater are prone to evaporation, whereas mineral oil and tape are not.The apparent temperature in each well was measured at fixed intervalsduring a 15-minute period.

[0108] The data show that evaporation affects the apparent temperatureof the samples. Specifically, the apparent temperature of wellscontaining water or alcohol was about 27° C., whereas the apparenttemperature of wells containing mineral oil or tape was about 29° C., orabout 2° C. warmer. Apparently, evaporation of water and alcohol coolsthe layer of gas above these fluids, leading to lower measuredtemperatures.

[0109] The data also show that evaporation affects the apparenttemperature stability of the samples. Specifically, after compensatingfor common-mode noise, wells containing water or alcohol showed about0.1° C. peak-to-peak (PTP) temperature variations, whereas wellscontaining mineral oil or tape showed about 0.01° C. PTP temperaturevariations, or about one-tenth as large. Thus, evaporation may precludeaccurate measurement of small thermal processes within a well containinga fluid prone to evaporation, at least if measured from above the well.Conversely, reducing evaporation may improve thermal signal and permit amore accurate measurement of thermal reactions within the well.

Example 4

[0110] This example describes results of an experiment designed todetermine whether temporal noise was caused by evaporation at thesurface of the fluid and whether temporal noise could be controlled byreading the fluid through a transparent film.

[0111] The experiment was performed using the top-read thermal-imagingapparatus and multiwell plate of Examples 2 and 3. However, here, eachwell was filled with water. Moreover, a first set of data was collectedas above, with the water exposed to ambient air, and a second set ofdata was collecting after placing a thin (˜0.0005-inch thick)infrared-transparent film (Poly IR II) over the wells, in direct contactwith the water to simulate a bottom-read design. The temperature in eachwell was again measured at fixed intervals during a 15-minute period.

[0112]FIG. 6A (“top read”) shows the range of measured temperatures as afunction of time, after subtraction of common-mode noise, reading fromthe surface of the exposed water. The data show significantthermodynamic noise, resulting from evaporation at the surface of thewater, with temperature variations of greater than about 0.1° C. (PTP).This noise level would make it very difficult to derive smalltemperature changes resulting from chemical or physiological processesbeneath the surface.

[0113]FIG. 6B (“bottom read”) shows the range of measured temperaturesas a function of time, after subtraction of common-mode noise, readingthrough the infrared-transparent film. The data show significantlyreduced temporal noise, with temperature variations of less than about0.025° C. (PTP). In this case, the film reduces or prevents evaporativecooling because the fluid no is longer exposed to dry air . Thisreduction in evaporative cooling reduces noise in the measurement, whichallows the system to record significantly smaller changes intemperature. This measurement technique, particularly when combined withthe novel multiwell plate design of FIGS. 2 and 3, allows accuraterecording of small subsurface processes taking place in the sample well,improving measurement resolution by about fourfold.

Example 5

[0114]FIG. 7 shows results of an experiment designed to determine thepreferred number of frames to average in the frame-averagingnoise-reduction technique. The experiment was performed using top-readthermal-imaging apparatus and low cross-talk multiwell plate of Examples2-4. The experiments show the noise level at several areas of themultiwell plate, averaged over 0, 2, 4, 8, and 16 frames at 60 Hz. Thefollowing chart summarizes the data for selected areas within the image:Normal 2× Frames 4× Frames 8× Frames 16× Frames Max (bit 10656.0010632.00 10632.00 10616.00 10600.00 counts) Min (bit 10560.00 10576.0010584.00 10576.00 10568.00 counts) Avg (bit 10611.17 10604.05 10610.2810592.24 10582.40 counts) Range 0.60 0.35 0.30 0.25 0.20 (Kelvin) StdDev 0.10 0.07 0.05 0.04 0.04 (Kelvin)

[0115] Values are in 14 bit units. The “std dev” row is theroot-mean-squared (RMS) noise of a uniform target after application offrame averaging.

Example 6

[0116]FIG. 8 shows results of an experiment designed to determine thelevel of common-mode noise typical of an infrared camera. Theexperiments were performed using the apparatus and plate of Examples2-5. The figure shows a typical raw data set after frame averaging(high-frequency noise reduction) and area averaging, but beforecommon-mode noise reduction and offset subtraction.

Example 7

[0117]FIG. 9 shows results of an experiment designed to assess theability of offset subtraction to extract data for a 135-μW reaction in amultiwell plate. The experiments were performed using the apparatus andplate of Examples 2-6.

[0118]FIG. 9A shows raw data for an experiment using four sample wellsin which one well received a “sample” comprising a constant 135-μWinput, while the other wells received a benign (i.e., non-reactive)sample. The data show the average temperature in the measurement regionas a function of time, after image averaging is applied.

[0119]FIG. 9B shows the same data after offset subtraction, whichadjusts the data so that each measurement starts at zero at time zero.There is a residual common-mode noise of approximately 0.05° C. PTP,after offset subtraction. This common-mode noise may be out of phase andmay shift depending on the geometric position of the cell, as shown inFIG. 8.

Example 8

[0120]FIG. 10 shows results of an experiment designed to assess theability of offset subtraction and the reduction of common-mode noiseusing a reference region local to the measurement region to extract datafor the 135-μW reaction of Example 7 and FIG. 9. The reduction ofcommon-mode noise reduces the noise level for the benign wells to an RMSlevel of about 0.004° C. The presence and thermal profile of the 135-μWreaction is clearly visible relative to the benign wells. The datacompare favorably to a thermal model that calculates the theoreticaleffect of a 135-μW input on a sample holder having the same fluidvolume. Without using the measurement and noise-reduction methodsdescribed here, the reaction resulting from a 135-μW input could not bedetected using an infrared camera.

Example 9

[0121] This example describes software for performing and/or evaluatingcalorimetric measurements.

[0122]FIG. 11 shows a software screen for the display of thermal data.The software screen may include one or more data presentation fields.These fields may be used to presentat data using any suitable form,including tables, graphs, and pseudo-images, among others. The fieldsmay include a single display that includes or summarizes data frommultiple samples, and/or multiple displays that each include orsummarize data from one or a subset of the multiple samples. If thereare multiple displays, they may be arranged in a manner representativeof the layout of the corresponding samples, such as an 8×12 array ofmini-graphs corresponding to the 8×12 array of samples in a standard96-well microplate. The data displayed in the software screens mayinclude a characteristic of the thermal radiation detected, such as theamount, intensity, and/or spectrum of the radiation. The data also mayinclude a computed quantity related to a characteristic of the thermalradiation detected, such as a temperature. The data also may includekinetic data, such as temperature versus time (denoted “tick”). Thesoftware screen also may include software switches for selecting thescale of the display, for example, X-axis and Y-axis scales forgraphical data and color schemes for pseudocolor images, among others.

[0123]FIGS. 12 and 13 show software screens for collecting, displaying,and/or calibrating data relating to the measurement and referenceregions. The top screen shows the application screen for collecting datausing the circular reference around the perimeter of the measurementregion. The bottom screen shot shows the setup window for defining thecharacteristics of the reference. These screens again may include one ormore data presentation fields and one or more software switches, amongothers. Moreover, these screens may permit recording and/or reporting ofsystem parameters, such as emissivity, object distance, ambienttemperature, relative humidity, noise reduction methods, and/or sampleholder format, among others.

Example 10

[0124] This example further describes noise-reduction methods providedby the invention.

[0125] The noise-reduction methods may include (1) converting detectedthermal infrared radiation to a signal, and (2) processing the signal toreduce the proportion of the signal that is attributable to noise. Thestep of processing the signal may include a step of temporally averagingthe signal comprising computing a quantity based on distinguishablecomponents of the signal representing thermal infrared radiationdetected from the same sample at different times. Alternatively, or inaddition, the step of processing the signal may include a step ofspatially averaging the signal comprising computing a quantity based ondistinguishable components of the signal representing thermal infraredradiation detected from different portions of the same sample.

[0126] The noise-reduction methods also may include (1) detectingthermal infrared radiation transmitted from a plurality of samplescontained in the sample wells using an optical device, (2) convertingthe thermal infrared radiation detected from each sample to acorresponding signal, and (3) adjusting the signals so that each has thesame preselected value at the same preselected time. The preselectedvalue may be zero, among others, and/or the preselected time may bezero, among others.

[0127] The noise-reduction methods also may include (1) detectingthermal infrared radiation transmitted from a reference region adjacenta sample, and (2) constructing a sample signal characteristic of thethermal infrared radiation detected from the sample based on the thermalinfrared radiation detected from the sample and the adjacent referenceregion. The reference region may comprise an annular portion of thesample plate distributed adjacent a perimeter and/or about a central oroptical axis of the sample well.

[0128] The disclosure set forth above encompasses multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious and directed to one of the inventions. These claims may referto “an” element or “a first” element or the equivalent thereof; suchclaims should be understood to include incorporation of one or more suchelements, neither requiring nor excluding two or more such elements.Inventions embodied in other combinations and subcombinations offeatures, functions, elements, and/or properties may be claimed throughamendment of the present claims or through presentation of new claims inthis or a related application. Such claims, whether directed to adifferent invention or to the same invention, and whether broader,narrower, equal, or different in scope to the original claims, also areregarded as included within the subject matter of the inventions of thepresent disclosure.

We claim:
 1. A plate for holding a plurality of samples, comprising: aframe; a plurality of sample wells disposed in the frame; and acorresponding plurality of thermal reference regions disposed adjacentthe sample wells in the frame; where the thermal reference regions havean emissivity of at least about 0.5; and where thermal infraredradiation detected from a sample positioned in at least one of thesample wells may be calibrated using thermal infrared radiation detectedfrom an adjacent thermal reference region.
 2. The plate of claim 1, theframe being substantially rectangular, where the length of the frameranges between about 125 mm and about 130 mm, and where the width of theframe ranges between about 80 mm and about 90 mm.
 3. The plate of claim1, where the number of sample wells in the plate is selected from thegroup consisting of 96, 384, 768, 1536, 3456, and
 9600. 4. The plate ofclaim 1, where the density of sample wells in the plate is at leastabout 1 well per 81 mm².
 5. The plate of claim 1, where the volume ofeach sample well in the plate is less than about 500 microliters.
 6. Theplate of claim 1, where the sample wells and the thermal referenceregions are composed at least in part of different materials.
 7. Theplate of claim 1, where the thermal reference regions have an emissivityof at least about 0.8.
 8. The plate of claim 1, where the thermalreference regions are positioned on a support structure, and where thethermal mass of the thermal reference regions and associated supportstructures is greater than the thermal mass of the sample wells andcorresponding samples.
 9. The plate of claim 1, where each thermalreference region includes an annular portion distributed adjacent aperimeter of a corresponding sample well.
 10. The plate of claim 1, thesample wells having a central axis, where each thermal reference regionincludes an annular portion positioned about the central axis of acorresponding sample well.
 11. The plate of claim 10, where the thermalreference regions are symmetric about the central axis.
 12. The plate ofclaim 1, the sample wells having a top, where each thermal referenceregion is positioned about or above the top of a corresponding samplewell.
 13. The plate of claim 1, the sample wells having a bottom, whereeach thermal reference region is positioned about or below the bottom ofa corresponding sample well.
 14. The plate of claim 1, where the samplewells and corresponding thermal reference regions are separated by a gapalong a line connecting each portion of the sample wells and thecorresponding thermal reference regions to reduce heat transfer betweenthe sample wells and the thermal reference regions.
 15. The plate ofclaim 14, where the gap is filled with air.
 16. The plate of claim 1,each thermal reference region having an emissive reference surface,where the emissive reference surface includes a metal.
 17. The plate ofclaim 16, where the metal is aluminum.
 18. The plate of claim 1, eachthermal reference region having an emissive reference surface, where theemissive reference surface is substantially flat.
 19. The plate of claim18, each sample well having a bottom, at least a portion of the bottombeing substantially flat, where the flat portion of the bottom of eachsample well is substantially parallel to the emissive reference surfaceof the corresponding thermal reference region.
 20. The plate of claim19, each sample well having a bottom, at least a portion of the bottombeing substantially flat, where the area of the flat portion of thebottom of each sample well is within a factor of ten of the area of theemissive reference surface of the corresponding thermal referenceregion.
 21. The plate of claim 1, where the thermal reference regionsare positioned at an end of a support member disposed between the samplewells.
 22. A plate for holding a plurality of samples, comprising: aframe; a plurality of sample wells disposed in the frame, the samplewells having a top and a bottom; and a corresponding plurality ofthermal reference regions disposed adjacent the sample wells in theframe; where the sample wells and corresponding thermal referenceregions are separated by a gap along a line connecting each portion ofthe sample wells and the corresponding thermal reference regions toreduce heat transfer between the sample wells and the thermal referenceregions; and where thermal infrared radiation detected from a samplepositioned in at least one of the sample wells may be calibrated usingthermal infrared radiation detected from an adjacent thermal referenceregion.
 23. The plate of claim 22, where the gap is filled with air. 24.The plate of claim 22, where the sample wells and the thermal referenceregions are composed at least in part of different materials.
 25. Theplate of claim 22, where the thermal reference regions have anemissivity of at least about 0.5.
 26. The plate of claim 22, where thethermal reference regions and associated support structure have a higherthermal mass than the sample wells and corresponding samples.
 27. Theplate of claim 22, the sample wells having a central axis, where eachthermal reference region includes an annular portion positioned aboutthe central axis of a corresponding sample well.
 28. The plate of claim22, the sample wells having a bottom, where each thermal referenceregion is positioned about or below the bottom of a corresponding samplewell.
 29. The plate of claim 22, each thermal reference region having anemissive reference surface, where the emissive reference surfaceincludes a metal.
 30. A plate for holding a plurality of samples,comprising: a sample holder having a plurality of sample wells; and athermal isolation member for supporting the sample holder so that eachsample well can be precisely positioned along an optical path, thethermal isolation member providing a thermally controlled thermalreference surface adjacent each well as viewed along the optical path.31. The plate of claim 30, where each reference surface defines anaperture framing the associated optical path.
 32. The plate of claim 30,where the sample wells and the thermal reference regions are composed atleast in part of different materials.
 33. The plate of claim 30, wherethe thermal reference regions have an emissivity of at least about 0.5.34. The plate of claim 30, where the thermal reference regions andassociated support structure have a higher thermal mass than the samplewells and corresponding samples.
 35. The plate of claim 30, the samplewells having a central axis, where each thermal reference regionincludes an annular portion positioned about the central axis of acorresponding sample well.
 36. The plate of claim 30, the sample wellshaving a bottom, where each thermal reference region is positioned aboutor below the bottom of a corresponding sample well.
 37. The plate ofclaim 30, where the sample wells and corresponding thermal referenceregions are separated by a gap along a line connecting each portion ofthe sample wells and the corresponding thermal reference regions toreduce heat transfer between the sample wells and thermal the referenceregions.
 38. The plate of claim 30, where each thermal reference surfacecomprises metal.
 39. A method of detecting thermal infrared radiation,comprising: providing a sample plate having a plurality of sample wellsand a corresponding plurality of thermal reference regions positionedadjacent the sample wells; providing an optical device configuredpreferentially to detect thermal infrared radiation; detecting thermalinfrared radiation transmitted from a sample positioned within at leastone of the sample wells using the optical device; detecting thermalinfrared radiation transmitted from a thermal reference regionpositioned adjacent the sample well holding the sample using the opticaldevice; and constructing a sample signal characteristic of the thermalinfrared radiation detected from the sample based on the thermalinfrared radiation detected from the sample and the adjacent thermalreference region.
 40. The method of claim 39, further comprisingcorrelating the detected radiation with the progress of a chemical orphysiological reaction occurring within the sample.
 41. The method ofclaim 39, the frame being substantially rectangular, where the length ofthe frame ranges between about 125 mm and about 130 mm, and where thewidth of the frame ranges between about 80 mm and about 90 mm.
 42. Themethod of claim 39, where the number of sample wells in the sample plateis selected from the group consisting of 96, 384, 768, 1536, 3456, and9600.
 43. The method of claim 39, where the density of sample wells inthe sample plate is at least about 1 well per 81 mm².
 44. The method ofclaim 39, where the volume of each sample well in the sample plate isless than about 500 microliters.
 45. The method of claim 39, where thesample wells and the thermal reference regions are composed at least inpart of different materials.
 46. The method of claim 39, where thethermal reference regions have an emissivity of at least about 0.5. 47.The method of claim 39, where the thermal reference regions andassociated support structure have a higher thermal mass than the samplewells and corresponding samples.
 48. The method of claim 39, the samplewells having a central axis, where each thermal reference regionincludes an annular portion positioned about the central axis of acorresponding sample well.
 49. The method of claim 48, the sample wellshaving a bottom, where each thermal reference region is positioned aboutor below the bottom of a corresponding sample well.
 50. The method ofclaim 39, where the sample wells and corresponding thermal referenceregions are separated by a gap along a line connecting each portion ofthe sample wells and the corresponding thermal reference regions toreduce heat transfer between the sample wells and thermal the referenceregions.
 51. The method of claim 39, each thermal reference regionhaving an emissive reference surface, where the emissive referencesurface includes a metal.
 52. The method of claim 39, where the step ofconstructing a sample signal includes the step of subtracting areference value from a corresponding measurement value, where thereference value is computed using the radiation detected from thethermal reference region, and where the measurement value is computedusing the radiation detected from the sample.
 53. The method of claim52, where the step of constructing a sample signal includes the step ofaveraging the thermal infrared radiation detected from at least twodifferent portions of the same sample.
 54. The method of claim 52, wherethe step of constructing a sample signal includes the step of averagingthe thermal infrared radiation detected from the same sample from atleast two different times.
 55. The method of claim 39, furthercomprising processing the sample signal to reduce the proportion of thesample signal that is attributable to noise.
 56. The method of claim 55,where the step of processing the sample signal includes the step ofcomputing a quantity based on distinguishable components of the samplesignal representing thermal infrared radiation detected from the samesample at different times.
 57. The method of claim 55, where the step ofprocessing the sample signal includes the step of computing a quantitybased on distinguishable components of the sample signal representingthermal infrared radiation detected from different portions of the samesample.
 58. The method of claim 39, further comprising performing thesteps of detecting thermal infrared radiation from a sample and anadjacent thermal reference region for a plurality of samples positionedin a corresponding plurality of sample wells in the sample plate. 59.The method of claim 58, further comprising: repeating the step ofconstructing a sample signal for the plurality of samples; and adjustingthe sample signals so that each has the same preselected value at thesame preselected relative time.
 60. The method of claim 59, where thepreselected value is zero.
 61. The method of claim 59, where thepreselected time is zero.
 62. The method of claim 58, where the thermalinfrared radiation is detected simultaneously from the plurality ofsamples.
 63. The method of claim 58, where the thermal infraredradiation is detected sequentially from the plurality of samples. 64.The method of claim 39, the sample plate comprising an insert portioncontaining the sample wells and a support portion containing the thermalreference regions, further comprising forming the sample plate by matingthe insert portion with the support portion.
 65. The method of claim 39,where the optical device comprises: an examination site; and a detectorconfigured to receive and preferentially to detect thermal infraredradiation transmitted from a sample positioned within a sample well atthe examination site.
 66. The method of claim 39, the sample wellshaving a central axis, the optical device having an optical axis,further comprising aligning the central axis and the optical axis priorto the steps of detecting thermal infrared radiation.
 67. The method ofclaim 39, further comprising shielding the sample from incidentradiation to reduce the proportion of the sample signal arising fromtransmission, reflection, and/or photoluminescence from the sample. 68.The method of claim 39, further comprising filtering the radiationtransmitted from the sample to extract thermal infrared radiation priorto the step of detecting thermal infrared radiation.
 69. The method ofclaim 39, where at least about half of the thermal infrared radiationdetected by the optical device has a wavelength between about 3micrometers and about 5 micrometers.
 70. The method of claim 39, whereat least about half of the thermal infrared radiation detected by theoptical device has a wavelength between about 7 micrometers and about 14micrometers.
 71. The method of claim 39, further comprising computing aquantity related to a characteristic of the thermal infrared radiationtransmitted from the sample.
 72. The method of claim 71, where thequantity is representative of the temperature of the sample.
 73. Themethod of claim 71, further comprising: computing the quantity for aplurality of samples; and displaying the quantities graphically in amanner representative of the arrangement of the corresponding samplewells in the sample plate.
 74. The method of claim 39, furthercomprising covering the sample wells to reduce evaporation from thesamples.
 75. A system for detecting thermal infrared radiation,comprising: a sample plate having a plurality of sample wells and acorresponding plurality of thermal reference regions positioned adjacentthe sample wells; an optical device configured preferentially to detectthermal infrared radiation transmitted from a sample positioned withinat least one of the sample wells and from an adjacent thermal referenceregion; and a processor configured to analyze the detected radiation andto construct a sample signal characteristic of the thermal infraredradiation detected from the sample based on the thermal infraredradiation detected from the sample and the adjacent thermal referenceregional.
 76. The system of claim 75, the sample plates having a centralaxis, the optical device having an optical axis along which radiationmay be detected, where the optical device supports the sample plate suchthat the central axis is aligned with the optical axis.